Disease-causing mutations of RhoGDIα induce Rac1 hyperactivation in podocytes

Abstract

Nephrotic syndrome (NS) describes a group of kidney disorders in which there is injury to podocyte cells, specialized cells within the kidney's glomerular filtration barrier, allowing proteins to leak into the urine. Three mutations in ARHGDIA, which encodes Rho GDP dissociation inhibitor α (GDIα), have been reported in patients with heritable NS and encode the following amino acid changes: ΔD185, R120X, and G173V. To investigate the impact of these mutations on podocyte function, endogenous GDIα was knocked-down in cultured podocytes by shRNA and then the cells were re-transfected with wild-type or mutant GDIα constructs. Among the 3 prototypical Rho-GTPases, Rac1 was markedly hyperactivated in podocytes with any of the 3 mutant forms of GDIα while the activation of RhoA and Cdc42 was modest and variable. All three mutant GDIα proteins resulted in slow podocyte motility, suggesting that podocytes are sensitive to the relative balance of Rho-GTPase activity. In ΔD185 podocytes, both random and directional movements were impaired and kymograph analysis of the leading edge showed increased protrusion and retraction of leading edge (phase switching). The mutant podocytes also showed impaired actin polymerization, smaller cell size, and increased cellular projections. In the developing kidney, GDIα expression increased as podocytes matured. Conversely, active Rac1 was detected only in immature, but not in mature, podocytes. The results indicate that GDIα has a critical role in suppressing Rac1 activity in mature podocytes, to prevent podocyte injury and nephrotic syndrome.

The FERM protein EPB41L5 regulates actomyosin contractility and focal adhesion formation to maintain the kidney filtration barrier

“…5). Although hyperactivation of Rac1 is an accepted pathogenetic model in podocyte disease verified by several independent studies (40,41), there is an ambiguous perception regarding the role of RhoA. Nevertheless, an emerging body of evidence supports the concept of a required balance of these GTPases to maintain cellular function (42).…”

ARF6 mediates nephrin tyrosine phosphorylation-induced podocyte cellular dynamics

“…Nephrin signaling-induced podocyte cytoskeletal remodeling is partially mediated by increased Rac1 activity and results in podocyte effacement [ 22 , 28 , 29 , 31 , 45 – 47 ]. In other cell models ARF6 modulates Rac1 activity effecting cellular architecture [ 36 , 37 , 39 , 48 , 49 ].…”

“…ARF-GEFs and -GAPs) [ 61 , 64 , 78 ]. For example, human gene mutations in Rac1 regulatory proteins, ARHGAP24 and ARGHDIA , have been reported to alter Rac1 activity and are associated with focal segmental glomerulosclerosis [ 12 , 29 – 31 ]; more so, recent evidence suggests that these disturbances are implicated in minimal change disease [ 12 ]. Identification of ARF6’s role in podocyte injury responses adds to this complexity.…”

“…Nephrin tyrosine phosphorylation-mediated increase in PI3K enzymatic activity subsequently increases activity of Rac1, a Rho family small GTPase that plays a pivotal role in membrane ruffling, cell motility, and actin organization [ 22 , 24 , 28 ]. Aberrations in Rac1 activity are associated with human minimal change disease and idiopathic focal segmental glomerulosclerosis [ 12 , 28 – 31 ].…”

Proteinuric Kidney Diseases: A Podocyte's Slit Diaphragm and Cytoskeleton Approach

“…However, these Rac1 knockout mice were also found to exacerbate the injury from chronic hypertensive glomerulosclerosis. Activation of Rac1 has shown to attenuate podocyte injury and expedite recovery in vitro ( 145 , 146 ), yet hyperactivation of Rac1 has been linked to diseased podocytes in a dose-dependent manner ( 147 – 149 ). For Cdc42, podocyte-specific deletion in mice led to early-onset severe proteinuria, FPE, glomerulosclerosis ( 144 ), and congenital nephropathy ( 150 ).…”